The term "Salmonella" as used herein, refers to the bacteria classified as such in Bergey's Manual of Systematic Bacteriology (N. R. Krieg [ed.], 1984, 427-458, Williams & Wilkins). Detection of Salmonella is important in various medical and public health contexts. The Salmonella species are important agents of human disease. Salmonella bacteria can cause a variety of pathological conditions ranging from simple gastroenteritis to more severe illnesses.
It is, therefore, an aspect of the present invention to provide a novel assay system capable of rapidly detecting Salmonella and which is generally applicable to environmental, food or clinical samples.
Pursuant to a standard laboratory method and a method recommended by the F.D.A. (FDA/BAM Bacteriological Analytical Manual, Chapter 7, 6th Edition, 1984, Supplement 9/87', Association of Official Analytical Chemists), the presence of Salmonella in environmental or food specimens has been traditionally detected by culturing an appropriately prepared sample on microbiological media under conditions favorable for growth of these organisms. The resulting colonies are then typically examined for morphological and biochemical characteristics, a process that generally is initiated 48 hours after acquisition of the sample and disadvantageously takes several days to complete.
It is another aspect of the present invention to avoid the disadvantage associated with traditional culturing techniques by providing more rapid methods to detect Salmonella.
Taber et al., U.S. Pat. No. 4,689,295, disclose the use of DNA probes specific for Salmonella DNA to detect the presence of bacteria of the genus Salmonella in food. There is generally, however, only one DNA copy per organism and thus a limited number of detectable "targets".
It is still yet another aspect of the present invention to provide probes and methods not limited to detecting DNA.
Ribosomes are of profound importance to all organisms because they serve as the only known means of translating genetic information into cellular proteins, the main structural and catalytic elements of life. A clear manifestation of this importance is the observation that all cells have ribosomes.
Ribosomes contain three distinct RNA molecules which, at least in E. coli, are referred to as 5S, 16S, and 23S rRNAs. These names historically are related to the size of the RNA molecules, as determined by sedimentation rate. In actuality, however, they vary substantially in size between organisms. Nonetheless, 5S, 16S, and 23S rRNA are commonly used as generic names for the homologous RNA molecules in any bacteria, and this convention will be continued herein.
Kohne et al. (1968) Biophysical Journal 8:1104-1118 discuss one method for preparing probes to rRNA sequences.
Pace and Campbell, Journal of Bacteriology 107:543-547 (1971), discuss the homology of ribosomal ribonucleic acids from diverse bacterial species and a hybridization method for quantitating such homology levels. Similarly, Sogin et al., Journal of Molecular Evolution 1:173-184 (1972), discuss the theoretical and practical aspects of using primary structural characterization of different ribosomal RNA molecules for evaluating phylogenetic relationships.
Fox et al., International Journal of Systematic Bacteriology (1977), discuss the comparative cataloging of 16S ribosomal RNAs as an approach to prokaryotic systematics. Hogan et al., International Patent Application publication number W088/03957, disclose a number of oligonucleotides that hybridize to some Salmonella rRNAs. None of these references, however, by themselves or in combination teach or predict the improved probes or methods of the present invention.
Lane, Rashtchian, and Parodos in copending U.S. Ser. No. 127,484 discloses a number of oligonucleotide probes for Salmonella rRNA and assay formats which enhance their utility. While the probes described therein work well, it is yet still another aspect of the present invention to provide further improved Salmonella-specific, rRNA targeted probes and probe sets and strategies for maximizing their utility.
As used herein, probe(s) refer to synthetic or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that allow them to hybridize under defined predetermined stringencies, specifically (i.e., preferentially) to target nucleic acid sequences.
Hybridization is traditionally understood as the process by which, under predetermined reaction conditions, two partially or completely complementary single-stranded nucleic acids are allowed to come together in an antiparallel fashion to form a double-stranded nucleic acid with specific and stable hydrogen bonds. The stringency of a particular set of hybridization conditions is defined by the base composition of the probe/target duplex, as well as by the level and geometry of mispairing between the two nucleic acids. Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and/or the temperature of hybridization. Generally, as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a corollary, the stringency of the conditions under which a hybridization is to take place (e.g., based on the type of assay to be performed) will largely dictate the preferred probes to be employed. Such relationships are well understood and can be readily manipulated by those skilled in the art. As a general matter dependent upon probe length, such persons understand stringent conditions to mean approximately 35.degree. C.-65.degree. C. in a salt solution of approximately 0.9 molar sodium chloride. A target nucleic acid sequence is one to which a particular probe is capable of preferentially hybridizing.
Still other useful definitions are given as their first use arises in the following text. All references cited herein are fully incorporated by reference.